The use of kits to detect certain Vibrio bacteria (sometimes referred to herein as Vibrio for brevity) in the environment, industrial settings, or on food is not a unique concept. U.S. Pat. No. 4,308,347 discloses a container device to detect pathogenic microorganisms in a fluid sample. The use of color generating chemicals was conceived as a mechanism to indicate the presence of pathogenic organisms with the intended result of a reduced need to use additional analysis equipment. U.S. Pat. No. 5,786,167 discloses a method to distinguish pathogen species such as those of Salmonella bacteria (sometimes referred to herein as Salmonella for brevity) by plating the sample on a solid medium of melibiose, mannitol, sorbitol, a pH indicator, and chromogenic substrate to reveal the presence of particular bacteria. If the test is positive, the bacteria is then cultivated and tested for additional indication for the presence of target pathogen specifically. U.S. Pat. No. 5,843,699 discloses a method of recognition and classification from a pre-enriched media that discourages the growth of non-target organisms while encouraging the growth of target microorganisms. The sample is then incubated and subjected to biochemical testing and analysis for results. To increase sensitivity, the prior art also discloses mechanisms to reduce the growth or generation of other bacteria within test samples, such as U.S. Pat. No. 5,208,150, which incorporates sodium salt to suppress the growth of competing organisms.
U.S. Pat. No. 6,136,554 discloses a method to detect E. coli, Salmonella, or a mixture of both by inoculating a nutrient medium into a sample and reviewing the color of bacterial colonies grown in the sample. If the result is negative, the process is repeated using a different biochemical tests for different bacteria.
Vibrio is the leading cause of food-borne illness from seafood consumption world-wide. Since 1996, a 400% increase in reported seafood-associated illnesses in the U.S. Current methods to detect and quantify Vibrio are so laborious and expensive that few laboratories in the world have analytical capabilities to produce reliable results, and the capacity at labs that do is very limited. More analytical resources are needed to monitor the coastal environment and seafood supply (e.g., surge resources in the event of an outbreak and mitigation resources for sample monitoring and validation of present post-harvest processing techniques). The prior art does not disclose a culture-based mechanism or kit to detect and quantify the presence and amount of pathogenic Vibrio at a high-sensitivity rate, that is more accurate, that significantly reduces the occurrence of Type-I or Type-II errors such as false-positives and false-negatives, that can be performed in a significantly reduced time period, that can be accurate and reproducible without the use of additional laboratory machinery or equipment, and that can be performed through a single-tube methodology.
Therefore, a need in the art for Vibrio assay kits with the previously mentioned attributes is evident.